Ultra-fast pathogen toxin detection assay based on microwave-accelerated metal-enhanced fluorescence

Abstract

The present invention provides for a system and method to detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, wherein the system uses a metal-enhanced fluorescence (MEF)-PA assay in combination with microwave-accelerated PA protein surface absorption. Microwave irradiation rapidly accelerates PA deposition onto the surface adjacent to deposited metallic particles and significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA.

That which is claimed is: 
     
       1. A method for detecting a target protein in a testing sample, the method comprising:
 a) providing a system comprising
 a substrate surface comprising immobilized metallic particles, 
 wherein the metallic particles have a diameter from about 40 nm to about 120 nm and separated from each other with a distance from about 10 nm to 50 nm; 
 
 b) introducing the testing sample to the substrate surface for a sufficient time and under conditions wherein the target protein in the testing sample binds to the substrate surface between the metallic particles and not to the metallic particles; 
 c) introducing a first antibody having affinity for the target protein under conditions sufficient for the binding of the first antibody to form a target protein/first antibody complex and applying microwave energy in an amount sufficient to increase the binding of the first antibody to the target protein; 
 d) introducing a second antibody having affinity for the first antibody, wherein the second antibody comprises at least one excitable molecule, wherein the excitable molecule is selected from the group of an intrinsic fluorophore, extrinsic fluorophore, and fluorescent dye, wherein binding to the first antibody positions the excitable molecule from about 5 nm to about 30 nm from at least one metallic particle; 
 e) applying electromagnetic energy in an amount sufficient to excite the excitable molecule; and 
 f) measuring the emissions from the excitable molecule and/or metallic particles to determine the existence of any target protein in the testing sample. 
 
     
     
       2. The method of  claim 1 , wherein the microwave energy is applied simultaneously during the introducing of the first antibody. 
     
     
       3. The method of  claim 1 , wherein the metallic particles have an elliptical, spherical, triangular or rod-like shape. 
     
     
       4. The method of  claim 1 , wherein the metallic particles are separated from each other by about 10 nm to about 30 nm. 
     
     
       5. The method of  claim 1 , wherein the excitable molecule is positioned from about 5 nm to about 20 nm from at least one metallic particle. 
     
     
       6. The method of  claim 1 , wherein the target protein in the testing sample is protective antigen PA83 of anthrax. 
     
     
       7. The method of  claim 1 , wherein the metallic particles are fabricated from Silver, Gold, Copper, Aluminum, Zinc, Nickel, Palladium, Tungsten, Platinum, Germanium, Indium, Iron, Tin, Rhodium or combinations thereof. 
     
     
       8. The method of  claim 1 , wherein the microwave radiation is applied in a frequency range from about 1 GHz to about 3 GHz with a power level in a range between about 100 watts and 700 watts.